The collecting duct plays a major role in the excretion of ammonium (NH3 and NH4+), the major regulatable component of urinary net acid excretion. The mechanism of ammonium transport in the collecting duct, however, is not fully understood. In collecting duct cells, NH4C1 addition causes a rapid cellular alkalinization from NH3 entry, followed by a slower acidification due to NH4+ entry. Transporters mediating this NH4+ flux and their physiological roles have not been determined. It is thought, however, that ammonium secretion by the collecting duct occurs by active proton secretion in parallel with the passive diffusion of NH3. This model is based on the dependence of ammonium secretion on luminal pH and on the low transepithelial NH4+ permeability which has been measured in the collecting duct. This low NH4+ permeability, however, reflects the permeability of the rate limiting membrane. Although transepithelial NH4+ permeability is low, permeability of a single plasma membrane could be the same or much higher than the transepithelial permeability. Thus, study of the mechanisms of direct NH4+ transport should be viewed as transport through two membranes in series which is the goal of this project. Unlike the more proximal collecting duct segments, in the terminal inner medullary collecting duct interstitial and luminal NH3 and NH4+ concentrations have been measured. Thus, transporter mediated NH4+ flux can be placed in context with known NH3 and NH4+ gradients to determine a possible physiological role for direct NH4+ transport. We will study the mechanism of NH4+ transport in the rat terminal inner medullary collecting duct (tIMCD). Proposal objectives are the following: 1) To identify transporters mediating NH4+ flux. 2) To determine the NH3 permeability and the relative NH4+ driven H+ flux across the apical and basolateral membrane. 3) To determine the relative contribution of these transporters to total transepithelial ammonia flux. 4) To determine if NH4+ provides a source of protons resulting in a measurable change in transepithelial total H+ flux. Objectives will be addressed initially with rat tIMCD cells in suspension and then confirmed and extended using the isolated perfused tubule technique. In vitro perfusion allows localization of transport processes to a specific plasma membrane.